Periodontal cell migration into the apical pulp.pdf

Journal of Oral Rehabilitation, 1993, Volume 20, pages 637-652

Periodontal cell migration into the apical pulpduring the repair process after pulpectomy inimmature teeth: an autoradiographic studyO. V O J I N O V I C and J. V O J I N O V I C Clinics for Children and Preventive Dentistry,Faculty of Stomatology, University of Belgrade

SummaryThe migration of dental papilla cells into the periodontium during the process ofroot development may occur as part of the process involved in the formation of theperiodontal tissues. The question posed is whether such cells under pathological con-ditions could retromigrate from periodontium into dental pulp and together with otherapical pulp cells of immature teeth, take part in the production of additional dentaltissue, e.g. 1) the tertiary/dentine under deep carious lesion where odontoblasts hadbeen destroyed 2) the dentine bridge on an amputation wound and 3) calcified tissuewhich closes an apex during the apexification process in immature teeth.

The migration of periodontal cells locally marked by H^ Thymidine immediatelyafter partial pulpectomy in immature dog's teeth* was analysed at observation periodsof 2, 24 and 50 h and also without H^ Thymidine labelling of periodontal cells 8 weeksafter pulpectomy. The marked cells were found in the early observation periods afterpulpectomy just in the places where the hard tissues were formed in the later obser-vation period of 8 weeks. They were found in large numbers just around the coagulatednecrotic foci. The finding supports the assumption that firm necrotic masses are a veryimportant stimulative factor in the reparation process in pulp and periodontium. Theexperiment also corroborated the existence of periodontal cell retromigration intoapical dental papilla of immature teeth. Future research should assess the possiblerole of the pathological condition in the determination of undifferentiated odontogenicectomesenchymal periodontal cells into odontoblasts.

IntroductionThe process of physiological cellular differentiation of odontogenic cells is determinedby a genetically conditioned succession of biological interactions (Ede, 1978; Wessels,1977). Exogenous factors can influence this process, as well as the formative activityof the cells, changing them both. Thus, dentine tissue produced by odontoblasts underthe conditions of defence and reparation are different in structure from that producedunder the normal circumstances of development i.e. tertiary dentine under deepcarious lesions, the dentine bridge under the amputation wound, the clacified conglom-erate in the apex formed during the apexification process of immature teeth (Seltzer &

* All these experiments followed the guidelines proposed by the Ethical Committee of the LA.S.P.

Correspondence: Professor Dr O. Vojinovie, Stomatoloski Fakultet, Klinika Za Decju 1 PeventivnuStomatologiju, 11000 Beograd, Serbia. . ...^ . ,; , ;

637

638 O. Vojinovie and J. Vojinovie ^ - . ;

Bender, 1984; Ten Cate, 1985; Vojinovie, 1974, 1977, 1987; Vojinovie & Srnic, 1975.There are two possible explanations of this phenomenon:

(i) pathological activity of the formative cells affect the structure of the tissuesproduced by the cells;

(ii) incompletely differentiated newly mobilized cells which are to substitute thedestroyed ones, produce dental tissues incomplete in structure.

If the explanation under (ii) is acceptable, the first question to be posed is whetherthe newly differentiated cells, which produce all the above mentioned pathologicaldentine tissues, belong to the cellular clones strictly determined just for dentine orwhether the precursors genetically predetermined for the other dental tissues couldalso be involved. The second question is whether these reparative cell precursors areeither localised in the neighbourhood of the destroyed odontoblasts or migrate out ofsome other part of the dental germ or tooth, changing under new conditions, theirgenetically predetermined differentiation course.

Numerous authors have pointed to the mutual ectomesenchymal origin of the cellsof the dental papilla, periodontium and the neighbouring alveolar one. Therefore, itseems logical to assume that they also have the mutual cell precursors (Johnstone &Listgarten, 1972; Thesleff & Hurmerinta, 1981; Ten Cate, 1969; Bernick & Grand,1982).

During the process of root development, the migration of the cells of a dentalpapilla into a dental follicle and biological interaction between these two kinds of cellsmay occur. Yoshikawa & KoUar (1981) pointed to the possibility that the cells of thedental papilla under physiological conditions, may take part in the formation ofperiodontium. Osborn in 1984 and Osborn & Price 1988, concluded that the cells ofthe dental papilla probably migrate into the dental follicle and that they, together withthe cells of the investing layer are responsible for the formation of the attachmentalveolar bone and gingival mesoderm. Others (Palmer & Lumsden, 1987; McCullochet al, 1987) have pointed to physiological migration of cells out of endosteal spacesinto periodontium and through vascular canals.

These kinds of cells influence the organization of the periodontal cellular populationand have effect on the cementum produced over the nearby root surface. The ex-periments described above indicate that the genetically harmonised interaction betweenodontogenic precursors of dental papilla, periodontium and nearby bone, is necessaryfor normal root development. Schroder (1973, 1985), investigated the effect of calciumhydroxide ions upon the formation of a dentine bridge over an amputation would haveafter pulpectomy. He concluded that the multilayer necrosis resulted in effect fromcalcium hydroxide ions during the period up to 24h. The author thought that the layerof firm necrosis closest to the pulp, mineralizing itself by attraction of salts, as wellas the layer of new odontoblasts just under the dentine bridge (this means under thepreviously formed and mineralized firm necrosis layer), raises the question of theodontoblast origin, as well as of their determination pathway.

There is little data available concerning cellular interactions in the pappiloper-iodontal ligament interface under pathological conditions. For apexification andapexogenesis after endodontic treatment of immature teeth the stimulative procedureis very important to introduce the exact route of the reparatory cells. The possibilityof retromigration of the papilla cells (those which, according to Yoshikava and Kollar(1981) and Osborn (1984) and Osborn and Price (1988), physiologically migrate fromthe papilla into dental follicle) from periodontium into papilla, could be proof of the

Periodontal cell migration 639

existence of undetermined dentinogenic cells in periodontal ligament and their positiverole in the reparative process in the apical pulp of immature teeth.

The aim of our experiments was to investigate, by local application of cellularmarkers in the already formed part of the apical periodontium in immature dog'steeth, the following: 1) whether the periodontal cells retromigrate after pulpectomyout of the periodontium into the dental papilla and 2) whether they take part in thefurther process of dentinogenesis during the formation of the apex.

Materials and methodsTwelve two rooted premolars (24 roots) of beagle dogs aged 67 months were used inthe experiment (Table 1). The contralateral teeth of the same jaw were used as acontrol. The experiment was performed right after the eruption of the teeth at thetime when radiologically no more than a third of the root was formed. The extirpationof the pulp out of all the premolars was done under rubber dam using broaches(0.4 mm wide) of a working length which corresponded to the formed part of a rootmeasured according to the radiograph. Attempts were made to remove the pulp outof one canal up to the level of apical opening but out of the canal of the other root ofthe same tooth up to the level 2 mm shorter than the apical opening (Scheme lA).Bleeding was stopped by a dry cotton pellet. Rinsing was done only with sterile salinesolution and under slight pressure. The canals were dried carefully and filled withCa(OH)2 paste prepared immediately before the application and avoiding mechanicalinjury of the tissue wound.

Sterile calcium hydroxide paste was prepared with distilled water without anyadditional medicaments. After the canal had been filled, it was closed with phosphatecement liner and contoured amalgam filling (Scheme lA). Just after the endodontictreatment described above, one drop of H'' Thymidine was applied by a syringe with0.45 mm lumen needle and shorter than the apical dentine edges. The length of needlepenetrating into the periodontium was controlled by X-ray (Scheme IB). In this way,no more than 1 microcurie of H'^ Thymidine was injected. The injection was repeatedon the other pulp extirpated premolars of the same dog on the second premolars.

Table 1. The number of teeth used in the experiment

The kind ofteeth

TH-^ applicatedobservation

period2h

24 h

50 h

TH"^ nonapplicated8 weeks

Total

I prem.

3 teeth(6 roots)

1 tooth(2 roots)4 teeth

(8 roots)

II prem.

3 teeth(6 roots)


(8 roots)

III prem.

3 teeth(6 roots)


(8 roots)

total

3 teeth(6 roots)3 teeth

(6 roots)3 teeth

(6 roots)

3 teeth(6 roots)12 teeth

(24 roots)

640 O. Vojinovic and J. Vojiriovicafter 22 h and on the third ones after 24 h. The dogs were sacrificed 2h after the lastexperiment. The observation periods of 2, 24 and 50h were obtained on each animalin this way.

On the three premolars of another dog the same experiment was performed with-out the application of H'^ Thymidine. This dog was sacrificed after 8 weeks (Table 1).The experiments were done under general anaesthetic by intravenous barbiturateinjection. The dogs were sacrificed by giving large doses of anaesthetic and then 10%formalin was injected by perfusion right into the heart.

After the preparation and removal of the complete jaws, fixation in 4% neutralformalin was performed (formaldehyde solution 3740% 100 cc, aqua dist. 900 cc,NaH2, PO4, H2O 4g., Na2HPO4 6.5 g). The jaws remained fixed for 7 days and thenout into small blocks. Each block included one tooth and the neighbouring bone. Thefixation of these blocks was performed in the following 7 days, the demineralization inthe solution of 24% EDTA, pH 7.4 lasting about 60 days.

Following demineralization and paraffin embedding, serial mesiodistally directedbuccolingual cutting was performed. Every 15th of the 5 mm sections was placed onthe plate together with the section of the control tooth, covered with gelatin, followedby stripped emulsion (Kodak R-10), free of its gelatin liner. The plates coated in thisway were kept in the dark for 14 days at 4C. and developed with Kodak D-19. Thesections were stained with either H&E, Gomori or PAS technique.

ResultsPulpectomy could not be performed completely in either of the roots. In each, thefragile buccal apical dentine edge was broken (Figs 1, 6, 10). For the sake of betterspace orientation, during the anaylsis, the apex was divided into three regions (scheme2A-transversal; buccolingual section, 2B-sagital: mesiodistal section). The region TIincludes the apex area on the level of the amputation would along the whole medisodistaland buccolingual diameter. The region T2 encompassed the part of the root under theregion TI in the whole buccolingual diameter, but not in the whole mesiodistal one.It incorporated only the part of the root where the marker was applied, which meansonly the middle part of the root starting from the mesial towards the distal diameterof the tooth (scheme 2A, 2B). The region T3 includes part of the root mesially anddistally from the region T2, that is the part which is furthest from the place of appli-cation of the marker (scheme 2B), but under the region TI.

Observation period of 2hRegion TI. Amputation wound: There were haemorrhagic foci with oedema and micro-phages. Subodontoblastic layer: Haemorrhagic foci were visible in all the specimens.

Region T2. Subodontoblastic layer: There were foci of haemorrhage in this region onboth buccal and lingual sides. The buccal dentine wall was interrupted in spots andthe apical immature pulp communicated, in these places, directly with periodontium(Fig. Al). There were scarce macrophages localized in groups. The region of epi-thelium sheet: On the buccal side there were various sized areas of destruction. Onthe lingual side, it was preserved along the whole mesiodistal diameter (Fig. 1 AH).

Region T3. Subodontoblastic layer: Haemorrhagic foci were noticeable in places, buttnore rarely than in the region TI and T2. Epithelium sheet: It was well preserved on


BFig. 1. Observation period of 2h. Region T2, b, buceal side root; 1, lingual side of root; II, undamagedside of epithelial sheet (H&E form 4%, EDTA 23%). (A) Transversal buceolingual seetion of theroot at the region T2. At this plaee, pulp direetly eommunieates with periodontium (21, 85x). (B)Solitary marked cell found on the border of the damaged periodontium towards pulp. The placeswhere the marked cells were found are indicated by X and x on the part A (289x).

both lingual and buccal sides of the root.Localiszation of the marked cells: Only a fewmarked cells were found in the periodontium, as well as on the pulp border near to it.i.e. in places where dentine wall was broken (Fig.l A, x, X, IB).

Observation period of 24hThis period was characterized by the organization of the coagulum.

Region TI. On the amputation wound microphages were dominant. In comparisonwith the period of 2h, macrophages were considerably more numerous. Coagulatednecrotic masses (CN Masses) were found in large quantites next to dentine wallsright under the amputation wound. They were found also in small quantities on theamputation wound.

Region T2. Apical part of the pulp: Blood vessels gathered in masses especiallyaround the fractured dentine edge with initial formation of granulation tissue. Thelayer of regular, undamaged dentine within this region both on the buccal and lingual sidewas separated from CN masses by a darker line or space (Fig. 2, 1). Subodontoblasticregion: An eosinophilic band and even argyrophilic foci were located. In the middlepart of the pulp in this area monocytes were dominant. Lymphocytes were scarce.Epithelium sheet: This was destroyed on the buccal while it was preserved on thelingual.

Region T3. CN Masses were scarce and the odontoblast layer was mainly preserved.Localization of the marked cells: Between macrophages on the amputation wounditself in the region TI there were no marked cells. They were found next to or insideCN masses located along dentine walls of the region T2 (Figs 2 and 3).

642 O. Vojinovic and J. Vojinovic

Fig. 2. Observation period of 24h. Region T2. Solitary makred cell inside CN masses, whieh areseparated from dentine by dark line. (H&E, form 4%, EDTA 24%) (578x) M, marked cell; k, CNmasses; 1, bordering line between CN masses and dentine; D, dentine.

Fig. 3. Observation period of 24h. Region T2. One of rare groups of marked cells next to CN massesin subodontoblastic region. It is evident that odontoblasts are destroyed in this place. M, labelled cells;D, dentine. K, coagulation necrotic masses (CN masses). 1, bordering line between predentine andCN masses. (H&E. form 4%, EDTA 24%) (131x).

Observation period 50 h.The beginning of the reparative process in this period was evident.

Region TI. On the amputation wound itself, there were thickly packed layers of cells(Figs 4, M and 5, M) Macrophages were dominant, without lymphocytes. Cells werenumerous next to CN masses (Figs 4, 5, M), which were found next to dentine andseparated from it by a clear bordering line or space (Fig. 5).


Fig. 4. Observation period of 50h. Region TI, amputation wound. The labelled cells were condensedjust on amputation wound and in subodontoblastic region. They are evidently rarer in the middleof pulp (H&E, form 4%, EDTA 24%) (131x) K, canal of root; D, dentine; P, apical pulp; M,marked cells.

Fig. 5. Observation period of 50h. Region TI. Labelled cells next to dentine wall on amputationwound. (H&E, form 4%, EDTA 24%, 433()53x). K, canal of root; M, marked cells; C, CN massesnext to dentine (D).

Region T2. On the lingual side the epithelial sheet was preserved and the apexogenesis(the physiological formation of the apex) was evident (Fig. 6, N). On the buccal sidethe dentine edge was fractured. Owing to dentine absence at this place, the pulp wasin direct contact with peridontium, in which CN masses could be found in areas (Fig.


1

Fig. 6. Observation period of 50 h. Region T2. Apical part of pulp (H&E, form 4%, EDTA 24%,25-5X). p, apical pulp; o, bordering line of pulp towards damaged dentine apical edge; PR, periodontium;D, dentine; K, CN masses; N, undamaged lingual side of apex. Firm neerotic masses squared under 1represent the places on which new dentine tissue is going to be produeed in order to form new apiealdentine wall in the later observation period presented in the Fig. lOA, B, and indicated with the letterB; s, space between CN masses and dentine.

6, K). Along the border line between the pulp and periodontium, there were thicklypacked spindle-shaped cells (Fig. 7, M). The cells were densely grouped around andinside the CN masses (Fig. 9). Next to the dentine wall on the undamaged lingualside, there were also CN masses which were separated from dentine by a darkerborder line or space (Figs 8 and 9B, C,D)

Region T3. The epithelial sheet was preserved on both sides. In the centre of theapical pulp, there were no inflammatory cells in this region. The localisation of themarked cells: they were found in large numbers in both the pulp and in periodontium.In the region TI, they were found in the amputation wound, where the start of col-lagen formation could be seen in places (Figs 4 and 5). Next to CN masses, along thedentine in this region, they were thickly grouped (Fig. 5). In the deeper regions,towards the middle of pulp, the marked cells were more rarely found. The impressionwas obtained that they migrate towards the wound (Fig. 4).

In the region T2, beside CN masses in the subodontoblastic region, on both thebuccal and lingual side, the marked cells were found mostly where the odontoblastshad been destroyed. (Fig. 9B, C,D). They were distinguishable from the well preservedodontoblasts by their shape and size. A thick layer of the marked cells was found on


Fig. 7. Observation period of 50 h. Region T2. Labelled eells on bordering pulp line towards damageddentine edge; pr, periodontium; M, marked cells; k, CN masses (H&E, form 4%, EDTA 24%, 289x).Place location of cells is squared on Fig. 6 and indicated by No. 1. (D).

Fig. 8. Observation period of 50 h. Marked cells localized next to CN masses at dentine wall onlingual undamaged side. Note dense marked cells just in subodontoblastic layer. They are extremelyrate in pulp centre (H&E, form 4%. EDTA 24%, 131 x). M, marked cells; K, CN masses; D, dentine.

the border between the pulp and periodontium on the buccal side, especially aroundCN masses (Fig. 7). Generally, it should be emphasized that the marked cells werefound mainly around CN masses and always in groups (Figs 5,7,8 and 9). The local-ization of the marked cells around the epithelial sheet was attractive: They werefound next to a newly formed dentine wall on its pulp side. On the periodontal side,they were only found apically. In the apical opening, next to the pulp limiting mem-brane, scarce groups of marked cells with small quantities of collagen were sporadicallyobservable. A few marked cells were recorded in the bone tissue only in the vicinityof the place of the marker appHcation. : . .,;


Fig. 9. Observation period of 50 h. Note marked eells localized around CN masses situated inperiodontium and next to dentine walls. Region T2. (H&E, form 4%, EDTA 24%). K, CN masses;d, dentine; s, empty space between dentine and CN masses; m, marked cells; f, condensed eollagen;p, periodontium; v. pulp; L, dark line between dentine and CN masses. (A) Marked cells insideperiodontium (m), around condensed collagen (f), on buccal side of root (289x). (B) Marked eellsinside pulp (m) next to and inside CN masses (k) loeated next to dentine (d) (234x). (C) Marked cells(m) next to CN masses separated from dentine (d) with empty space (s). In the middle of pulp therewere no groups of marked cells (v) (131x). Buccal side of the root. (D) The lingual undamaged side.Numerous marked cells (m) next to CN masses in odontoblasts. Bordering line (1) between dentineand CN masses (k) is well noticeable (175x).

Observation period of 8 weeksThe apical part of the immature pulp did not show any inflammatory changes. Towardsthe root canal, as well as towards the part of periodontium where the apical edge hadbeen broken, unspecific dentine tissues was formed. This tissue separated the pulpfrom a new periodontium forming in this way, the buccal wall of a new apex (Figs 10A, B, lOBB). The layers of this tissue which were formed first, were of an irregularstructure i.e. towards the periodontium (Figs 10, AR, 10, BR.). The closer to the pulpthe more regular the dentine structure became (Figs WA "T", lOB 'T") . The layer ofodontoblasts was also noticeable in the pulp by this tissue (Fig lOA, "O" lOB, "O").A thin layer of predentine was also observed and therefore confirmed that regulardentinogenesis was still taking place within the 8 week period (Fig. lOB "T").

At the side where the epithelial sheet was preserved, the root formation con-tinued by the regular apexogenesis process. The root assumed an approximatelynormal shape (Fig 10A, lOB). In the periodontium, which had the normal form on the


Fig. 10. Pathological dentine tissue was formed just on the place where marked cells had beenlocalized in the observation period of 50h (H&E, form 4%, EDTA 24%). D, regularly formed lingualdentine wall; B, irregularly formed buccal dentine wall on the damaged side of root; C, dentine bridgetowarde canal lumen; O, odontoblasts; R, layer of atubullar dentine on damaged buccal dentine wall;P, pulp; T, layer of tubular dentine on damaged bueeal dentine wall; TD, tertiary dentine; N, bone;Ou canal lumen of root; pr, periodontium. (A) Reconstruction (25-5x). Observation period of 8weeks. Third mandibular molar. It should be noticed that apex has almost regular shape duringapexification process of 8 weeks. Buccal damaged side of root (B) eonsists of two layers; primarilyformed irregular (R) and secondary formed, towards pulp, regular tubular dentine (T), with neighbouringodonotoblasts (O). The first layer merges with the second one without a bordering line. (B) Observationperiod of 8 weeks; second premolar of the same dog presented in Fig A. Odontoblastic layer to newlyformed apical wall is discernible (O). Process of apxification is identical to the one presented in Fig. A(25-5X). (C) Observation period of 8 weeks. Dentine bridge on amputation wound. The same twolayers could be seen as on damaged buccal apical wall; irregular (R) and tubular dentine on pulpalside (T) (117-5X) TD Tertiary dentine. (D) Tertiary dentine of the human permanent teeth, whoseformation was indueed by the trauma during the drilling. Note a layers of atubullar dentine betweendentine layers fomed before and after the trauma (175x).

undamaged lingual side, there were no pathological changes (Fig. lOA, D, lOB, D).On the injured buccal side, the periodontium was larger and of irregular shape, but inthe observation period of 8 weeks, without any inflammatory cells (Fig. 10 Apr, 10Bpr). It is important to point out that the new reparation hard tissue was formed,after the observation period of 8 weeks, just in the places in which the labelled cells

648 O. Vojinovic and J. Vojinovicwere found accumulated in groups in the observation period of 50h. i.e. 1) On theamputation wound a dentine bridge was formed after 8 weeks (Figs 4 and 5 in com-parison with Fig 10A,C, 10 C). 2) Repair took place in the dentine root wall betweenthe pulp and periodontium in places where the dentine edge was broken (Figs 6 and 7in comparison with Fig 10 A,B 10 B,B). 3) Tertiary dentine was formed on theundamaged lingual side (Fig. lOA "TD" in comparison with Figs 8 and 9D). It wasinteresting to note that the whole new dentine wall was formed on the damaged buccalside after 8 weeks, which corresponed to the normal apical shape (Fig 10A B",lOB "B"). The dentine tissue of this wall was very similar to the one which formedthe barrier on the amputation wound (Fig. IOC), as well as the tertiary dentine underthe deep cavities (Fig. lOD "TD". Both of them were of dentine type structure andformed by odontoblasts.

DiscussionImmature teeth were used in the experiment because of the pronounced immaturity ofthe cell population inside the apical part of the dental papilla. This situation offeredan opportunity to analyse the mode in which the genetically predetermined directionof differentiation, migration and function of the periodontal cells was changed afterpulpectomy.

The marker was introduced locally (not intraperitoneally) in order to avoid thesimultaneous labelling of the dental papilla cells and to make the migration of essentiallyperiodontal cells observable. The observation periods of 2, 24 and 50h were supposedto offer data on the migration of periodontal cells immediately after trauma andbefore the moment the cells performed their ultimate determination.

The aim of the additional experiment on the dogs sacrificed after 8 weeks andwithout labelling the periodontal cells, was to find the places in the apical immaturepulp in which the described dentine reparative tissue was definitively formed. It alsoallowed comparison to be made with the places where the marked cells were foundin the earlier observation period of 50 h. The aim was to see whether the labelledperiodontal cells could take part in the formation of pathological dentine tissue. TheTubilitec liner avoided the contamination of the amputation wound with the inter-position of bacteria as an additional irritative factor in the determination process ofthe marked cells. Calcium hydroxide for the canal filling was used because it has beenestablished that it does not bring about any immunological reactions (Schroder, 1973,1985; Vojinovic, 1975).

The injuries of the fragile dentine edge, found always on the buccal side, could beexplained by the slope of the root, which directed the nerve broach towards thebuccal side. However, there is a slight possibility for the edge being broken from theperiodontal side, by the syringe needle during the marker application. The aboveconsiderations arise from the fact that the bone was not injured in that place, whereasa part of the apical dentine was missing (Figs 1,6), The position of fragments of theinjured dentine indicated that the injury occurred from the pulpal side and the pulptissue on the buccal side was injured, while the periodontium was spared (Fig. 6).The identical injury was noticed in the teeth analysed 8 weeks after the experiment inwhich Thymidine had not been applied (Fig. lOA, B). The identical injuries wereevident in all the other previous similar experiments (Vojinovic, 1974, 1975, 1986),which were carried out without Thymidine.

The interruption of dentinogenesis in the places of the most severe trauma was


evident already after 2 and 24 h. This is confirmed by the absence of odontoblasts inthe area and haemorrhagic foci and spindle-shaped cells next to the dentine wall.

The findings of only a few labelled cells in the close vicinity of the application placeof the markers after 2h and their abundance after 50 h is in accord with the findingsby Gould et al. (1980). Following work on rats they suggested that proliferativeactivity of the cells of the periodontium does not take place before 30 h. The raremarked cells, found after 2h, were probably fibroblasts which, at the moment of theapplication of the marker, were in the phase of physiological replication.

The most frequent localization of the marked periodontal cells next to CN masses,no matter where they were located (in the pulp or periodontium) indicates that thesecells, together with the others, take part in the organization of the CN masses andtheir later mineralization, as well as in the subsequent production of the mineralizedtissue around the same CN masses (Figs 2, 5, 7, 8 and 9).

The first layers of the dentine bridge on the amputation wound (Fig. lOA "C", lOB"C", IOC), the first layers of the tertiary dentine in the places where odontoblastswere destroyed (Fig. WA "CTD"), as well as the mineralized tissue which, in theprocess of apexogenesis, separated the apical pulp stump from the periodontium (inthe areas of the broken dentine edge. Fig. lOA "B", WB "B"), were of the similarirregular structure. This result points to possible identity of their formative cells. Themarked periodontium cells were grouped right in the places where the formationof these tissues was to be expected (Figs IA, 4, 5 and 7) and already after 50h.Doubtlessly, these cells are highly involved in the production of these tissues. At thebeginning of their activity, the cells (the CN foci having been organized) produced,at first, an untypical dentine which, in the course of the process of cell differentiation,becomes more and more similar to orthodentine (Fig. WA "B", WB "B", WC "TD",WD "TD").

This fact points to the assumption that the marked cells in this experiment, dis-regarding their periodontal origin, could represent the precursors of odontoblastsand, owing to pathological conditions, began their formative activity before theyreached their own histomorphologic perfection. Another possible role of these cellscould be some kind of 'helping clone' for the organized synthetic activity.

The marked cells next to the dentine wall were evidently different by their shapeand volume from normal odontoblasts (Fig. 9B, D). This was not the same with thecells in the periodontium (Fig. 9A). This also could be evidence of possible periodontalcell migration during the reparative process after pulpectomy in immature teeth.

The assumption that the marked cells inside the pulp tissue have not been markedby the migration of cells but by the diffusion of the marker cannot be accepted for thefollowing reasons: 1) In the observation period of 2h, the presence of marked cellswas unnoticed in the apical opening, although this region was rich in cells in the divisionstage at the time of apex formation. 2) Marked cells were found mostly in groups andin the places where the formation of the dentine tissue was expected to take place(Figs 4, 5, 7, 8 and 9); if the diffusion was to be the reason, the marked cells should bedispersed all over the pulp. 3) The marked cells were found mostly round the CNmasses or inside them, and in the areas where the production of collagen was expectedto take place (the regions TI, T2, around the dentine fragments, on the border line ofthe pulp towards the periodontium (Fig. 7). 4) They were rare on the periodontal sideof the apical edge, which would not have been logical if the cells had been marked bythe marker diffusion, because in that periodontal region there were abundant immature

650 O. Vojinovic and J. Vojinoviccells. 5) In the centre of the pulp, there were a few marked cells, but they grew denserand denser towards the amputation wound, which indicates the migration of the cellsrather than the marker diffusion (Figs 4, 8 and 9C). 6) In the periodontium, coronal tothe place of the marker application, there were no marked cells in comparison with thenumber inside the pulp. The expectation would have been to find the cells in the perio-dontium rather than in the pulp since it would be easier for diffusion straight throughthe periodontium than into the pulp around the dentine edge. In fact they were foundin the odontoblast layer both on the side of application (Fig. 9C) and on the oppositeside (Fig. 9D), but just a few could be seen in the middle of the pulp (Fig. 8), i.e.between these two mentioned labelled layers.

Three questions arise from the reported experiments: 1) The meaning of thedifferentiation of periodontal cells into odontoblasts, taking into the considerationthat they genetically have another formative task. 2) To explain the presence of theodontoblast precursors in the periodontium. 3) To determine which inductive factorsprovide the conditions for the process of differentiation of periodontal cells intoodontoblasts.

If the assumption is acceptable that the marked periodontal cells are the precursorsof the new odontoblasts, besides other pulp cells, then it should be supposed that inthe periodontium of immature teeth, there are such precursors which are formativelymultipotent. This means that their final determination depends not only on the geneticinformation, but also on the outside conditions under whose influence the determinationtakes place. The differentiation of these precursors into odontoblasts can happen onlyif the cells have the appropriate pulp localization. Taking into consideration Yoshikawaand KoUar's (1981) findings together with those of Osborn (1984) and Osborn and Price(1988) that the dental papilla cells migrate into the dental follicle under physiologicalconditions, it could be assumed that the same cells could retromigrate into the apicalpulp tissue under pathological conditions. The second question posed above could beexplained through an intercommunication of undifferentiated pulp cells and periodontalcells which is possible through the wide apical opening.

The initiative factors for changing the direction of the cells which possible retro-migrate from the periodontium, could be various including inflammation of amputationwound, mechanical factors and broken dentine fragments. Within the ground substance,special initiative factors could also exist, especially in the process of separating thedental pulp from the periodontium (Fig. 6 and 10A) (Vojinovic et al., 1986). Havingin mind that the labelled cells were found mainly around or inside the CN masses,they also could be one of initiative factors.

Naturally at present the explanation concerning the initiation factors which inducedthe migration of cells can be accepted only as a supposition to be given further con-sideration. The reported experiments are clinically significant in that they recommendthat during the endodontic treatment of immature teeth, proper care should be givento the periodontium so as to enable it to perform its activity as physiologically aspossible. Besides, the fragile apical dentine edges are often exposed to fracture bypulpectomy. Therefore, in immature teeth, pulpectomy should be done with measuredneedles a millimetre shorter than the dentine wall length. Conditions should beprovided in the apical part of the pulp and periodontium for the above mentioneddifferentiation of reparatory cells and their undisturbed interactions in the pulpo-periodontal apical region.


Conclusions :The retromigration of the cells from the periodontium into the apical pulp stump afterpulpectomy in immature teeth is possible, provided that the cells have not fullydifferentiated. These cells are probably the same ones which migrated under thephysiological conditions of dentine root development, from the dental papilla into theperiodontium in order to take part in the formation of the attachment apparatus. Thispoints to the multipotentiality of the pulpo-periodontal cells in the apical part ofimmature teeth. Having come again into the dental papilla, under the pathologicalconditions after pulpectomy, it is not impossible that they differentiate into odon-toblasts. These experiments demonstrate that firm necrotic foci have some coordinatingeffect on this cellular activity. This is supported by their localization in those placeswhere the formation of the reparation dentine as well as the multitude of markedperiodontal cells around them are expected (amputation wound, dentine wall, miner-alized barrier between the apical pulp and periodontal ligament). Therefore, pulpectomyin immature teeth should be done in a way which enables the intercommunication inthe pulpoperiodontal cellular population. This implies the preservation of the apicalpulp, with the utmost care given to the prevention of the injury or loading of theapical part of the immature periodontium.

AcknowledgmentThis research work has been supported by the Scientific Fund of The Republic ofSerbia No. 1315 h.

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